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Homogeneously developed oak (Quercus robur L.) microcuttings were challenged in a Petri-dish system with the mycobionts Piloderma croceum J. Erikss. & Hjortst. and Paxillus involutus (Batsch) Fr. Non-destructive observations over 10 wk followed by d. wt measurements at the end of the assays served to precisely characterize root and shoot development, dynamics of mycorrhizal colonization and morphological ratio. In the system, plant development, and especially root morphogenesis, had more similarities to those of stump cuttings or of older seedlings than to those of 3-month-old seedlings. Whereas Paxillus involutus displayed early mycorrhizal colonization and had no significant morphological effects on the host Piloderma croceum modified markedly the entire plant development before a delayed mycorrhiza formation. The latter mycobiont stimulated elongation and production of the lateral root system and also increased the leaf surface. However, no corresponding weight increases were noted, which was reflected by significant increase of both specific root length and specific leaf area. These differential effects are discussed in relation to data concerning carbon requirement and auxin production of the mycobionts. The developed system was shown to be highly suitable for comparative studies with diverse mycobionts on recognition and physiological balance between partners before, and in the early stage of, formation of mycorrhizas.

Research on mycorrhizal interactions has traditionally developed into separate disciplines addressing different organizational levels. This separation has led to an incomplete understanding of mycorrhizal functioning. Integration of mycorrhiza research at different scales is needed to understand the mechanisms underlying the context dependency of mycorrhizal associations, and to use mycorrhizae for solving environmental issues. Here, we provide a road map for the integration of mycorrhiza research into a unique framework that spans genes to ecosystems. Using two key topics, we identify parallels in mycorrhiza research at different organizational levels. Based on two current projects, we show how scientific integration creates synergies, and discuss future directions. Only by overcoming disciplinary boundaries, we will achieve a more comprehensive understanding of the functioning of mycorrhizal associations., All authors acknowledge funding from the German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig funded by the German Research Foundation (DFG;FZT118) .A.M.M.,A.B.,M.P.,S.R., and N.M.v.D.acknowledge stimulating discussions within COSTActionFA1405 and funding from EUMC-ITNMiRA(grantno.765290).I.C.M. acknowledges financial supportfromtheDFG(grantno.ME4156/2-1)and Volkswagen Foundation [grantno.11-76251- 99-34/13 (ZN2928)].M.C.R.acknowledgesfundingfromtheEuropeanResearchCouncil(ERC,AdvancedGrant ‘Gradual Change’) andBMBFfundingfortheproject ‘Bridging inBiodiversityScience(BIBS)’. M.P.acknowledgessupportfrom MINECO (grantno.AGL2015-64990-C2-1-R).I.F.acknowledgessupportfromiDivFlexpoolProgram(grantno.RA-185/ 17). O.F.andN.E.acknowledgesupportfromtheERC(EuropeanUnion’s Horizon2020researchandinnovationprogram, grant no.677232).S.R.acknowledgestheSwissScienceFoundation(grantno.159869).F.B.,S.H.,andM.T.acknowl- edge DFGfor financial support(GrantsBU941/20-1andTA290/4-1).AlltheauthorsacknowledgesupportfromtheiDiv Open SciencePublicationFund.

As long-lived organisms, trees use resources to support both growth and below- and aboveground trophic interactions. Resources fluctuate in relation to periods of growth cease that are regulated by internal and external factors, and these fluctuations feed backs to trophic partners. Some major forest trees display an endogenous growth rhythm, and related pulses of variation in allocation of resources have been detected. As this trait makes it possible to separate growth into defined phases, it offers an opportunity to disentangle the intermingled complex regulation of growth and multitrophic interactions in trees. We present “TrophinOak”, a platform using microcuttings of pedunculated oak, a tree that displays endogenous rhythmic growth characterized by alternating shoot and root growth flushes. We select seven beneficial or detrimental above- and belowground partners including animals (Lymantria dispar, Pratylenchus penetrans, Protaphorura armata), fungi (Piloderma croceum, Microsphaera alphitoides, Phytophthora quercina) and bacteria (Streptomyces sp.), to synthesize bi- and tripartite trophic interactions, including ectomycorrhizal symbioses, and monitor fluctuations of carbon and nitrogen allocation as well as plant gene expression at distinct phases of oak growth. We use this model to identify and resolve the experimental challenges inherent in synthesizing diverse types of associations in a common microcosm system, in labeling plants with stable N and C isotopes and in analyzing transcripts in a non-model plant, a process which requires generating a specific contig library. We develop hypotheses and experimental design to test them in order to identify core mechanisms that help trees to modulate their own development and their multitrophic interactions for optimizing their long term performance in their environment. First results constitute a proof of concept that the platform works and enables us to test the hypotheses.

Highlight Increased resource availability has no impact on endogenous rhythmic growth and related allocation shifts in oak trees; an internal clock regulates core genes during shoot and root growth cessation phases., Common oak trees display endogenous rhythmic growth with alternating shoot and root flushes. To explore the mechanisms involved, microcuttings of the Quercus robur L. clone DF159 were used for 13C/15N labelling in combination with RNA sequencing (RNASeq) transcript profiling of shoots and roots. The effect of plant internal resource availability on the rhythmic growth of the cuttings was tested through inoculation with the ectomycorrhizal fungus Piloderma croceum. Shoot and root flushes were related to parallel shifts in above- and below-ground C and, to a lesser extent, N allocation. Increased plant internal resource availability by P. croceum inoculation with enhanced plant growth affected neither the rhythmic growth nor the associated resource allocation patterns. Two shifts in transcript abundance were identified during root and shoot growth cessation, and most concerned genes were down-regulated. Inoculation with P. croceum suppressed these transcript shifts in roots, but not in shoots. To identify core processes governing the rhythmic growth, functions [Gene Ontology (GO) terms] of the genes differentially expressed during the growth cessation in both leaves and roots of non-inoculated plants and leaves of P. croceum-inoculated plants were examined. Besides genes related to resource acquisition and cell development, which might reflect rather than trigger rhythmic growth, genes involved in signalling and/or regulated by the circadian clock were identified. The results indicate that rhythmic growth involves dramatic oscillations in plant metabolism and gene regulation between below- and above-ground parts. Ectomycorrhizal symbiosis may play a previously unsuspected role in smoothing these oscillations without modifying the rhythmic growth pattern.

Summary The root-rot pathogen Phytophthora quercina is a key determinant of oak decline in Europe. The susceptibility of pedunculate oak (Quercus robur) to this pathogen has been hypothesized to depend on the carbon availability in roots as an essential resource for defense. Microcuttings of Q. robur undergo an alternating rhythm of root and shoot growth. Inoculation of mycorrhizal (Piloderma croceum) and nonmycorrhizal oak roots with P. quercina was performed during both growth phases, that is, root flush (RF) and shoot flush (SF). Photosynthetic and morphological responses as well as concentrations of nonstructural carbohydrates (NSC) were analyzed. Infection success was quantified by the presence of pathogen DNA in roots. Concentrations of NSC in roots depended on the alternating root/shoot growth rhythm, being high and low during RF and SF, respectively. Infection success was high during RF and low during SF, resulting in a significantly positive correlation between pathogen DNA and NSC concentration in roots, contrary to the hypothesis. The alternating growth of roots and shoots plays a crucial role for the susceptibility of lateral roots to the pathogen. NSC availability in oak roots has to be considered as a benchmark for susceptibility rather than resistance against P. quercina.

Summary Oaks (Quercus spp.), which are major forest trees in the northern hemisphere, host many biotic interactions, but molecular investigation of these interactions is limited by fragmentary genome data. To date, only 75 oak expressed sequence tags (ESTs) have been characterized in ectomycorrhizal (EM) symbioses. We synthesized seven beneficial and detrimental biotic interactions between microorganisms and animals and a clone (DF159) of Quercus robur. Sixteen 454 and eight Illumina cDNA libraries from leaves and roots were prepared and merged to establish a reference for RNASeq transcriptomic analysis of oak EMs with Piloderma croceum. Using the Mimicking Intelligent Read Assembly (MIRA) and Trinity assembler, the OakContigDF159.1 hybrid assembly, containing 65 712 contigs with a mean length of 1003 bp, was constructed, giving broad coverage of metabolic pathways. This allowed us to identify 3018 oak contigs that were differentially expressed in EMs, with genes encoding proline-rich cell wall proteins and ethylene signalling-related transcription factors showing up-regulation while auxin and defence-related genes were down-regulated. In addition to the first report of remorin expression in EMs, the extensive coverage provided by the study permitted detection of differential regulation within large gene families (nitrogen, phosphorus and sugar transporters, aquaporins). This might indicate specific mechanisms of genome regulation in oak EMs compared with other trees.

Ectomycorrhiza, a symbiosis between soil fungi and the rootlets of major forest trees, is characterized by well defined anatomical traits but also encompasses a wide range of ecological and physiological situations. Functional studies of this symbiosis therefore address different kinds of systems. Here we review works done on an experimental model with micropropagated oak cuttings infected in a Petri dish system with the basidiomycote Piloderma croceum. The model is characterized by a high demand for carbohydrates by the fungus and the only differentiating of mycorrhizas with plants having a sufficient carrying capacity in terms of photoassimilate production. Already during the pre-mycorrhizal stage symbiotic interactions between the partners are observed at the morphogenetic and physiological levels and are influenced by the typical endogenous rhythmic development of the plant with alternating growth flushes in the shoot and in the roots. The system was used for first molecular and transcriptomic studies based on a subtractive suppressive hybridization, a macro-array experiment and the research for specific genes.

International audience; Expression of two plant chitinase genes, representing members of class III chitinases, was studied in Quercus robur roots during interactions in a pre-mycorrhizal stage with the ectomycorrhizal fungus Piloderma croceum. Chitinase gene expression was compared in lateral roots destined to form ectomycorrhiza, and in principal roots that are not directly involved in mycorrhizal interactions. The transcript level of the first chitinase (QrchitIII-1) was upregulated in lateral roots, whereas no significant differential expression was observed in principal roots. The second chitinase (QrchitIII-2) was regulated neither in lateral nor in principal roots in presence of the fungus. Because P. croceum did not induce significant chitinase responses in principal roots, the enhanced expression of QrchitIII-1 in lateral roots after inoculation may be related to some steps in symbiosis ontogenesis.

Summary • An in vitro system with micropropagated oaks ( Quercus robur ) and the ectomycorrhizal fungus Piloderma croceum , which is characterized by a delayed mycorrhiza formation, was used to identify plant transcripts upregulated in the premycorrhizal phase. • Complementary DNA (cDNA) populations of uninoculated roots and fungal mycelium were subtracted from a cDNA population of inoculated roots. Differential expression was confirmed by reverse Northern and 50 clones for different polypeptides were found to be up-regulated. Twenty-nine clones were investigated in more detail. • For approximately half of the cDNA fragments no homologies could be identified in databases. The residual fragments code for polypeptides with homologies to known proteins involved in signal perception and transmission, stress responses, metabolism and growth. • Since many of the identified genes have not yet been described in the context of symbiotic events, their potential roles during early phases of the recognition process are discussed.

The growth kinetics of Dactylis glomerata following a root excision were studied in order to characterize the process of partitioning of carbon assimilates between shoots and roots. Plants were grown hydroponically, in constant environments. The variations with time of shoot and root fresh weights during regrowth were measured using a nondestructive method. The effect of root excision on shoot and root water contents was also determined from complementary experiments. Immediately after cutting, the specific growth rate increased markedly for roots and decreased slightly for shoots, but this decrease can be at least partly accounted for by the variation of the shoot water content. At the end of the perturbed phase, the specific growth rates and the root fraction had almost returned to the values measured immediately before cutting. The experimental regrowth kinetics were analysed in terms of a kinetic model which calculates shoot and root growth as the difference between the carbon provided by photosynthesi s and carbon losses associated with respiration. A satisfactory description of the regrowth kinetics was obtained by assuming that the partitioning of carbon substrates between shoots and roots is not modified by cutting, and that the partitioning coefficient remains constant during regrowth. According to this analysis, the stimulation of root growth after cutting can be mainly attributed to the reduction in the part of carbon substrates utilized in roots for maintenance requirements.

This study describes and analyses the regrowth kinetics of vegetative plants of Dactylis glomerata L. following defoliation. Plants were grown hydroponically in growth chambers at two constant temperatures, 17 and 25 °C. The variations with time of shoot and root f. wts after the cutting were measured using a non-destructive method. During regrowth, the shoots were found to grow almost exponentially with time, whereas the growth rate of roots slowed markedly. A decrease in root f. wt was observed under the severest level of cutting. The regrowth kinetics were analyzed on an energetical basis. Shoot and root growth were calculated as the difference between the input of the carbon substrates supplied by photosynthesis and the loss of carbon associated with respiratory processes. A satisfactory description of the experimental kinetics was obtained under the main assumption that during regrowth the net photosynthetic production is partitioned between shoots and roots according to a constant ratio. From this analysis, the effect of defoliation can be interpreted in terms of a perturbation of the internal energetical equilibrium which, in roots, modifies the proportion of carbon substrates utilized for growth and maintenance requirements.

The formation of the ectomycorrhiza implies an alteration in gene expression of both the plant and fungal partners, a process which starts before the formation of any symbiotic interface. However, little is known on the regulation pattern occurring in different parts of the root system. Our experimental system consisting of a micropropagated oak with a hierarchical root system was shown to exhibit symbiosis functional traits prior to any mycorrhizal tissue differentiation after the inoculation with the basidiomycete Piloderma croceum. Using a cDNA array, the plant gene regulation was analyzed in the pre-mycorrhizal phase. Seventy-five transcripts showed differential expression in pre-mycorrhizal lateral and principal roots, and both root types exhibited different sets of responsive genes. For transcripts selected according to a statistical analysis, the alteration in gene expression was confirmed by RT-PCR and quantitative real-time PCR. Genes regulated in pre-mycorrhizal lateral roots displayed an almost identical expression in mycorrhizas. In contrast, genes regulated in pre-mycorrhizal principal roots were often regulated differently in ectomycorrhizas. Down-regulation affected most of the regulated genes involved in metabolism, whereas most of the regulated genes related to cell rescue functions, water regulation and defence response were up-regulated. Regulation of such genes could explain the increase of global resistance observed in mycorrhizal plants.

A Petri dish system in which development of oak (Quercus robur L.) microcuttings is stimulated by the late stage ectomycorrhizal (EM) fungus Piloderma croceum J. Erikss. & Hjortst. in a long pre-symbiotic stage was optimised to allow synchronous, rhythmic plant growth. Addition of indole-3-acetic acid or activated charcoal to the medium caused an early and more intensive EM formation coupled with suppression of most developmental effects of P. croceum. Leaf area, chlorophyll fluorescence, and content were compared in inoculated and uninoculated plants grown at two relative humidity levels (45 and 95%) and under consideration of three possible answers to inoculation, that is, no or EM formation after the 1st or the 2nd shoot flush. The culture conditions for uninoculated plants were suboptimal, leading toward photochemical stress reflected by a non photochemical quenching (qE) increase and a reduced Chl content at the end of the assay. Prior to EM formation, inoculation itself enhanced the optimal (Fv/Fm) and effective (phiPSII) quantum yield in leaves of the 1st shoot flush under reduced relative humidity. It also fully protected the plants against stress during the complete assays. The results indicate that inoculated plants only form EM once they have acquired a sufficient development level and C-providing capacity. However, the fungus actively improves the development and photosynthesis of plants up to the pre-mycorrhizal stage, helping them to reach this capacity.